WO2023063738A1 - Mesenchymal stem cell-derived extracellular vesicles to which anti-ace2 antibody is attached, and use thereof - Google Patents

Abstract

The present invention relates to mesenchymal stem cell-derived extracellular vesicles to which an anti-ACE2 antibody is attached, and use thereof. More specifically, it has been identified, from the results of extracting extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) in which an anti-ACE2 antibody is expressed on the surface thereof and investigating the effectiveness of the extracellular vesicles to the entry of pseudo-virions, that the mesenchymal stem cell-derived extracellular vesicles to which the anti-ACE2 antibody is attached of the present invention can prevent the infection of SARS-CoV2 by blocking ACE2, and thus is expected to be used effectively as an agent for blocking early stage infection of COVID-19.

Description

Anti-ACE2 antibody-derived mesenchymal stem cell-derived extracellular vesicles and uses thereof

The present invention relates to mesenchymal stem cell-derived extracellular vesicles to which anti-ACE2 antibodies are attached and uses thereof.

Coronavirus is a virus composed of a single piece of the (+) strand RNA genome of about 27-32 kb, distributed in humans and other mammals. It is known that coronaviruses produce 6 to 8 subgenomic RNAs that have a common mRNA at the 3' end of genome-RNA through replication and transcription processes. In most people, coronavirus infection causes mild symptoms but is highly contagious, with SARS (Severe Respiratory Syndrome, 10% mortality rate) coronavirus and MERS (Middle East Respiratory Syndrome, 37% mortality) coronavirus affecting more than 10,000 people over the past 20 years. has been infected

COVID-19, a recently discovered new coronavirus (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) infection, was discovered in China on December 1, 2019 and first reported on December 12, 2019 as an acute respiratory syndrome. , symptoms include fever, cough, shortness of breath, and atypical pneumonia. Since January 2020, it has spread widely outside of China, and it is developing into a situation of concern, such as a rapid transmission around the Lunar New Year holiday in China, a rapid increase in the number of infected people, and the paralysis of the entire city of Wuhan.

SARS-CoV2 binds to the angiotensin-converting enzyme 2 (hACE2) protein, which is mainly present in human lung epithelial cells, invades cells and reproduces the virus by replicating its genetic material inside human cells. Finding strategies to prevent the coronavirus from entering human cells is a fundamental way to treat the COVID-19 pandemic. The spike protein S1-RBD (Receptor Binding Domain) present on the surface of the corona virus binds to the hACE2 protein, and the three-dimensional structure of the binding at this time has been revealed by recent studies.

If the combination of SARS-CoV2 and hACE2 can be blocked, it is expected to fundamentally prevent the cell invasion of coronavirus, and the combination of SARS-CoV2 and hACE2 to prevent or treat infections caused by SARS-CoV2, COVID-19. Research on new means to block

An object of the present invention is to provide mesenchymal stem cell-derived extracellular vesicles (EV) expressing an anti-ACE2 antibody or an antigen-binding fragment thereof.

Another object of the present invention is an antiviral composition for coronavirus containing the extracellular vesicles as an active ingredient, a pharmaceutical composition for preventing or treating coronavirus infection, or for preventing or treating acute respiratory distress syndrome (ARDS) It is to provide a pharmaceutical composition.

In order to achieve the above object, the present invention is a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1; And mesenchymal stem cell-derived extracellular vesicles (EVs) expressing an anti-ACE2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 are provided.

In addition, the present invention provides an antiviral composition for coronavirus comprising the extracellular vesicles as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles as an active ingredient.

The present invention relates to an anti-ACE2 antibody-derived mesenchymal stem cell-derived extracellular vesicle and its use, and more specifically, to mesenchymal stem cell (MSC) expressing an anti-ACE2 antibody on its surface. As a result of extracting extracellular vesicles (EVs) from cells and verifying the effect of the extracellular vesicles on Pseudo-Virion entry, mesenchymal stem cell-derived extracellular vesicles attached with the anti-ACE2 antibody of the present invention Since it was confirmed that SARS-CoV2 infection can be prevented by blocking ACE2, it is expected to be useful as an infection blocker in the early stage of coronavirus infection.

Figure 1 shows the results of the virus infection blocking test analysis of extracellular vesicles (Extracellular vesicle; EV).

2 shows the results of regeneration analysis (MTS) of cells damaged by LPS.

Figure 3 shows the results of regeneration analysis (cell count) of cells damaged by LPS.

Figure 4 shows the results of regeneration analysis (Trans-well permeability assay) of cells damaged by LPS.

The present invention comprises a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1; And mesenchymal stem cell-derived extracellular vesicles (EVs) expressing an anti-ACE2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2 are provided.

Preferably, the mesenchymal stem cells may express the anti-ACE2 antibody or antigen-binding fragment thereof on the surface through lentivirus infection, but is not limited thereto.

The "anti-ACE2 antibody" of the present invention is an antibody (C3 antibody) confirmed to bind to ACE2 in Korean Patent Application No. 10-2021-0130808 previously filed by the present inventors, and the amino acid sequence of the C3 antibody is shown in the table below. Same as 1.

antibody type		amino acid sequence	sequence number
C3	light chain	QSVLTQPPSVSGAPGQRVTISCTGSSSNIGADYDVHWYQQLPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEANYYCQSYESGLGWLFGGGTQLTVL	One
	heavy chain	QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYGRGLPPGHFDYWGQGTLVTVSS	2

The "extracellular vesicle (EV)" of the present invention refers to nano-sized vesicles derived from cells, and depending on the secretion type and size, exosomes, microvesicles, and ectosomes ), microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles. Extracellular endoplasmic reticulum is a major means of communication between cells, including nucleic acids and proteins, which are the main components of cells.

As used herein, the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes, for example, monoclonal antibodies, Clonal antibodies, full-length antibodies and antibody fragments may all be included. Also, the term “antibody” may include bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies or tetrabodies.

As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof comprising a variable region VH and three constant regions CH1, CH2 and CH3 comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen. can include all In addition, as used herein, the term “light chain” may include both a full-length light chain and fragments thereof including a variable region VL and a constant region CL, which include an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen. there is.

In the present invention, the terms "fragment", "antibody fragment" and "antigen-binding fragment" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv, and the like.

In addition, the present invention provides an antiviral composition for coronavirus comprising the extracellular vesicles as an active ingredient.

Preferably, the coronavirus may be SARS-CoV2, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles as an active ingredient.

Preferably, the coronavirus infection may be COVID-19, but is not limited thereto.

In the present invention, "COVID-19" refers to a novel coronavirus infection, and represents variants of SARS and MERS as RNA viruses. COVID-19 shares about 77.5% sequence identity with SARS and about 50% with MERS. However, in contrast to SARS and MERS, the spike glycoprotein of COVID-19 forms a structure in which one RBD domain protrudes upward, which causes the target receptor ACE2 (angiotensin) and It shows 100 to 1,000 times stronger binding force. This strong binding force makes it easier to penetrate into cells, thereby increasing the infectivity.

In addition, the present invention provides a pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles as an active ingredient.

Preferably, the ARDS may be due to a coronavirus infection, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is one commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, and starch. , acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear including, but not limited to, acid magnesium and mineral oil, and the like. The composition for preventing or treating cancer metastasis of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. can be administered with When administered orally, since proteins or peptides are digested, oral compositions can be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention can be used in any device through which the active agent can move to target cells. can be administered by

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

< Example 1> extracellular endoplasmic reticulum ( Extracellular vesicle; EV) viral infection blocking test analysis

A lentivirus was prepared by transferring the Sc-Fv DNA sequence of an antibody (C3 antibody) confirmed to bind to ACE2 in Korean Patent Application No. 10-2021-0130808 previously filed by the present inventors into a lentivirus vector. This lentivius was infected with mesenchymal stem cells (MSC), and as a result, C3 antibody was expressed on its surface. Afterwards, EVs were extracted from the antibody-expressed mesenchymal stem cells. To verify the effect of EV on pseudo-virion entry, HEK-293T-hACE2 cells (NR-52511, BEI resources) were treated with various concentrations of EV (0ng/ml, 0.5ng/ml, 5ug/ml). . In addition, SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit (NR-52948, BEI resources) were infected. Cells were lysed at 48 hpi and Luciferase activity was measured with Bright-Glo™ (E2610, Promega, Madison, WI, USA) according to the manufacturer's instructions. Quantification of Luciferase activity was performed using a Perkinelmer EnVision microplate reader. The protocol of the experiment is described in Crawford, KHD, et al. "Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays." Viruses 12 (ZOZOI: E513. PubMed: 32384820). As a result of the experiment, virus infection was reduced when antibody-expressing EVs were treated compared to control EVs (MSC-EVs without antibody expression) (FIG. 1). As a result of the above experiment, it means that the EV of the present invention can prevent infection of SARS-CoV2 through ACE2 blocking.

< Example 2> with LPS damage Mouth cell regeneration assay (MTS)

Cell experiments using MTS were conducted to confirm that the efficacy of MSC-EVs to regenerate cells damaged by LPS due to cell engineering was not impaired. Cells used for the assay were Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150), seeded in a 96 well plate, and cultured for 24 h. Thereafter, LPS (Sigma, #L2880) was pretreated with 1 ug/ml for 1 hour, and EVs were treated with 5 ng/ml concentration. Afterwards, in order to check the viability of the cells at 24, 48, and 96 hpt, a reagent (Promega, CellTiter 96® AQueous One Solution Cell Proliferation Assay, G3582) was treated, followed by a 2-hour reaction (37°C, 5% CO ₂ ), Absorbance was measured at 470 nm. As a result, it was confirmed that the group treated with the EV of the present invention restored cell viability to a similar extent to that of the control EV (FIG. 2).

< Example 3> with LPS damage Analysis of regeneration of worn-out cells (cell count)

In order to confirm that the efficacy of MSC-EVs to regenerate cells damaged by LPS due to cell engineering was not impaired, an experiment was conducted to measure cell count. Cells used in the assay were Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150), seeded in a 6 well plate, and cultured for 24 h. Thereafter, LPS (Sigma, #L2880) was pretreated with 1 ug/ml for 1 hour, and EVs were treated with 5 ng/ml concentration. Thereafter, trypsin-EDTA was treated to confirm cell proliferation at 24, 48, and 96 hpt, and cell numbers were measured. As a result, it was confirmed that the group treated with the EV of the present invention restored cell proliferation to a similar degree to that of the control EV (FIG. 3).

< Example 4> with LPS damage Trans-well permeability assay

In order to confirm that the efficacy of MSC-EVs to regenerate cells damaged by LPS due to cell engineering was not impaired, an experiment was conducted to measure trans-well permeability. Cells used for the assay are Human Bronchial Epithelial Cell Line (16HBE14o-, Sigma. SSC150), seeded on collagen coated Insert (Falcon®, Permeable Support for 24-well Plate with 0.4 μm Transparent PET Membrane, #353095) ) and incubated for 48h-72h with the bottom plate until a monolayer was formed. Thereafter, LPS (Sigma, #L2880) was pretreated with 1 ug/ml for 1 hour, and EVs were treated with 5 ng/ml concentration. Then, in order to measure the permeability of cells at 24 hpt, FITC-Dextran (Sigma, #53379) was treated and reacted (15 min, RT), and then the basal media of the bottom plate was measured at (em/ex 485 nm/530 nm) wavelengths did As a result, it was confirmed that the group treated with the EV of the present invention restored the cells to a similar degree to the control EV and reduced the permeability of the cell layer (FIG. 4).

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

1. A light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1; and mesenchymal stem cell-derived extracellular vesicles (EVs) expressing an anti-ACE2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 2.
2. The extracellular vesicles according to claim 1, wherein the mesenchymal stem cells express the anti-ACE2 antibody or antigen-binding fragment thereof on the surface through infection with a lentivirus.
3. An antiviral composition against coronavirus comprising the extracellular vesicles of claim 1 or 2 as an active ingredient.
4. The antiviral composition according to claim 3, wherein the coronavirus is SARS-CoV2.
5. A pharmaceutical composition for preventing or treating coronavirus infection comprising the extracellular vesicles of claim 1 or 2 as an active ingredient.
6. The pharmaceutical composition for preventing or treating coronavirus infection according to claim 5, wherein the coronavirus infection is COVID-19.
7. A pharmaceutical composition for preventing or treating acute respiratory distress syndrome (ARDS) comprising the extracellular vesicles of claim 1 or 2 as an active ingredient.
8. The pharmaceutical composition for preventing or treating ARDS according to claim 7, wherein the ARDS is caused by a coronavirus infection.

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